Photodynamic therapy composition

ABSTRACT

A polymerizable phthalocyanine compound having the formula (I) or a PSMA-targeted phthalocyanine compound conjugate thereof having the formula (V).

RELATED APPLICATION

This application is a Continuation-in-Part of U.S. Ser. No. 16/901,874,filed Jun. 15, 2020, which is a Continuation-in-Part of U.S. Ser. No.16/279,326, filed Feb. 19, 2019, which is a Continuation of U.S. Ser.No. 15/629,281, filed Jun. 21, 2017, (now U.S. Pat. No. 10,207,005),which is a Continuation-in-Part of U.S. Ser. No. 14/767,984, filed Aug.14, 2015, (Now U.S. Pat. No. 9,889,199), which is a National Phase ofPCT/US2014/016932, filed Feb. 18, 2014, which claims priority to U.S.Provisional Application Ser. No. 61/765,346, filed Feb. 15, 2013. Thisapplication also claims priority from U.S. Provisional Application No.62/861,802, filed Jun. 14, 2019, the subject matter of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

This application relates to diagnostic and therapeutic compounds andcompositions, and more particularly relates to phthalocyanine compoundsand their use in targeted photodynamic therapeutic compositions for thetreatment and detection of cancer.

BACKGROUND

Photodynamic therapy, hereinafter also referred to as “PDT”, is aprocess for treating cancer wherein visible light is used to activate asubstance, such as a dye or drug, which then attacks the tumor tissuethrough one or more photochemical reactions, thereby producing acell-killing, or cytotoxic, effect. When certain photosensitizercompounds are applied to a human or animal body, they are selectivelyretained by cancerous tissue while being eliminated by healthy tissue.The tumor or cancerous tissue containing the photosensitizer can then beexposed to therapeutic light of an appropriate wavelength and at aspecific intensity for activation. The light energy and thephotosensitizer cause a photochemical reaction that kills the cells inwhich the photosensitizer resides.

Phthalocyanines, hereinafter also abbreviated as “Pcs”, are a group ofphotosensitizer compounds having the phthalocyanine ring system.Phthalocyanines are azaporphyrins consisting of four benzoindole groupsconnected by nitrogen bridges in a 16-membered ring of alternatingcarbon and nitrogen atoms (i.e., C₃₂H₁₆N₈) which form stable chelateswith metal and metalloid cations. In these compounds, the ring center isoccupied by a metal ion (either a diamagnetic or a paramagnetic ion)that may, depending on the ion, carry one or two ligands. In addition,the ring periphery may be either unsubstituted or substituted. Thesynthesis and use of a wide variety of phthalocyanines in photodynamictherapy is described in International Publication WO 2005/099689.Phthalocyanines strongly absorb clinically useful red or near IRradiation with absorption peaks falling between about 600 and 810 nm,which potentially allows deep tissue penetration by the light.

PDT agents such as Pc4, a silicon phthalocyanine photosensitizer areapproved by the FDA for clinical trials. However, due to its highlyhydrophobic nature, Pc4 is insoluble in aqueous media and thus is oftenrequires formulation into delivery vehicles to be useful. In addition,it is also difficult to functionalize the terminal tertiary amine Pc4 atthe axial position of the molecule. Therefore, there remains a need formore functional PDT agents.

SUMMARY

Embodiments described herein relate to polymerizable phthalocyaninecompounds and PSMA-targeted conjugates thereof for use in the detectionand treatment of PSMA expressing cancers and particularly for use inmethods of treating PSMA expressing cancers using photodynamic therapy(PDT). The polymerizable phthalocyanine compounds are based on analogsof the PDT photosensitizing compound Pc4. The compounds feature amethacrylate moiety which readily allows for polymerization as well asmaking the compounds available for additional chemistries afforded bythe terminal double bond. The polymerizable phthalocyanine compoundsdescribed herein have been found to be effective as a small moleculealternative to Pc4 in targeted imaging and/or targeted PDT of cancer ina subject where they can provide a more stable therapeutic PSMA-targetedcomposition.

Some embodiments described herein relate to a polymerizablephthalocyanine compound having the formula (I):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and

R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, carboxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio, C₁₋₆hydroxyalkyl, C₁₋₆alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In other embodiments, the PSMA-targeted phthalocyanine compound can havethe formula (II):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and

R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, carboxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio, C₁₋₆hydroxyalkyl, C₁₋₆alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In still other embodiments, the PSMA-targeted phthalocyanine compoundcan have the formula (III):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and

R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, carboxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio, C₁₋₆hydroxyalkyl, C₁₋₆alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In certain embodiments, the PSMA-targeted phthalocyanine compound hasthe formula (IV):

or a pharmaceutically acceptable salt thereof.

Other embodiments described herein relate to a pharmaceuticalcomposition for treating cancer in a subject. The composition includes aplurality of PSMA-targeted phthalocyanine compound conjugates having theformula (V):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5, Y is a PSMA targeting moiety having a terminalcysteine residue, and wherein the PSMA targeting moiety targets thecomposition to a PSMA expressing cancer cell.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In certain embodiments, the PSMA-targeted phthalocyanine compoundconjugates have the formula (VI):

or a pharmaceutically acceptable salt thereof;

wherein Y is a PSMA targeting moiety having a terminal cysteine residue,and wherein the PSMA targeting moiety targets the composition to a PSMAexpressing cancer cell.

In particular embodiments, the PSMA-targeted phthalocyanine compoundconjugates have the formula (VII):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the PSMA targeted phthalocyanine compound isformulated in a pharmaceutically acceptable carrier. In someembodiments, the composition is formulated for systemic administration.

Other embodiments described herein relate to a method of treating PSMAexpressing cancer in a subject. The method includes administering to asubject with PSMA expressing cancer a therapeutically effective amountof a pharmaceutical composition.

The pharmaceutical composition includes a plurality of PSMA-targetedphthalocyanine compound conjugates having the formula (V):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5, Y is a PSMA targeting moiety having a terminalcysteine residue, and wherein the PSMA targeting moiety targets thecomposition to a PSMA expressing cancer cell.

The method also includes detecting the PSMA-targeted phthalocyaninecompound conjugates bound to and/or complexed with the PSMA expressingcancer cells to determine the location and/or distribution of the cancercells in the subject and irradiating the detected PSMA-targetedphthalocyanine compound conjugates, thereby inducing the cytotoxiceffects of the phthalocyanine compound on the cancer cells.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In some embodiments, the method further includes the steps of surgicallyresecting the cancer in the subject, wherein the detected PSMA-targetedphthalocyanine compound conjugates bound to and/or complexed with thecancer cells guide surgical resection of the cancer, and irradiating thePSMA-targeted phthalocyanine compound at the site of surgical resection,thereby inducing the cytotoxic effects of the phthalocyanine compound onresidual cancer cells following surgical resection.

In some embodiments, the surgical resection site is irradiated with anamount of radiation effective to inhibit tumor recurrence in thesubject. In some embodiments, intra-operative imaging (IOI) of thePSMA-targeted phthalocyanine compound conjugate bound to and/orcomplexed with the cancer cells can be used to define a tumor margin inthe subject to guide surgical resection of the cancer.

In some embodiments, the PSMA-targeted phthalocyanine compound conjugatecan have the formula (VI):

or a pharmaceutically acceptable salt thereof;

wherein Y is a PSMA targeting moiety having a terminal cysteine residue,and wherein the PSMA targeting moiety targets the composition to a PSMAexpressing cancer cell.

In certain embodiments, the PSMA expressing cancer is selected from thegroup consisting of renal carcinoma, transitional cell carcinoma of theurinary bladder, testicular embryonal carcinoma, colonic adenocarcinoma,neuroendocrine carcinoma, gliobastoma multiforme, malignant melanoma,pancreatic ductal carcinoma, non-small cell lung carcinoma, soft tissuecarcinoma, breast carcinoma, and prostatic adenocarcinoma. In particularembodiments, the cancer is metastatic prostate cancer.

In some embodiments, the pharmaceutical composition is administeredsystemically. Systemic administration can include intravenous injection.In some embodiments, the pharmaceutical composition is formulated in apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings which are presentedfor the purpose of illustrating the invention and not for the purpose oflimiting them.

FIGS. 1 (A-B) illustrate the experimental design and generation of ROSin vivo. (A) Scheme of experimental design. (B) Detection of ROS in vivoafter PDT. Mice bearing PC3pip tumor received PSMA-1-Pc413 and 24 hourslater were administered ROSstar800cw, which detects ROS. BothPSMA-1-Pc413 and ROSstar800cw fluorescence were measured before andafter light irradiation. Fluorescent signal in the deep red channel wasobserved after PDT, indicating generation of ROS after PDT.

FIGS. 2 (A-B) illustrate in vivo fluorescence imaging of mice bearingorthotopic PC3pipGFP tumor. (A) Representative images of whole mouse,primary tumor, and LNs. PSMA-1-Pc413 was able to detect both primarytumor and LN metastasis. White arrow, iliac LNs. Representative imagesare shown from five animals. (B) Histologic analysis of resected primarytumor and LNs. Presence of tumor cells in LNs was confirmed by GFPsignal, PSMA-1-PC413 signal, and hematoxylin and eosin (H&E) staining.White asterisks (*), the rim of lymphocytes in LNs. Red arrows anddashed blue out lines indicate the residual lymphocyte islands in LNsurrounded by tumor cells. Images in orange boxes are the enlargedmicroscopic images of the rectangles in column 1.

FIGS. 3 (A-D) illustrate the use of PSMA-1-Pc413 for IGS and PDT. (A)Representative images of WLS mice under Maestro and Curadel imagingsystems. Circles, surgical bed. (B) Representative images of IGS+PDTmice under Maestro and Curadel imaging systems. Minimal amount of GFPand PSMA-1-Pc413 signals was observed in the wound with loss ofPSMA-1-Pc413 signal due to photo activation. Circles, surgical bed. (C)Quantification of GFP signals in the three experimental groups beforesurgery (left), after surgery (middle), and after IGS+PDT (right).Before surgery, similar GFP signal was observed in three experimentalgroups before surgery (left). After surgery, significantly lower GFPsignal was observed in the IGS and IGS+PDT groups than in the WLS group(*, P<0.05:middle). Values are mean±SD (n=5 animals for WLS and IGS; n=8for IGS+PDT). (D) Quantification of PSMA-1-Pc413 signals in theexperimental groups before surgery (left), after surgery (middle), andafter IGS+PDT (right). After surgery, a significant difference wasobserved between IGS/IGS+PDT and WLS group (middle;*, P>0.05). Withinthe IGS+PDT group, PDT further reduced PSMA-1-Pc413 signal significantlyas compared with after IGS alone (right). Values are mean±SD (n=5 forWLS and IGS, n=8 for IGS+PDT).

FIGS. 4 (A-B) illustrate a combination of IGS and PDT delayed tumorprogression recurrence and extended animal survival. (A) Representativepostsurgery monitoring images of mice from WLS, IGS, and IGS+PDT groupsmeasured using Maestro GFP channel. (B) Tumor recurrence curves of micefrom three experimental groups (n=animal numbers). IGS did notsignificantly delay tumor recurrence as compared with WLS (P=0.2222).The tumor recurrence was significantly delayed by IGS+PDT. *, P=0.0008,IGS+PDT vs. WLS; #, P=0.00084, IGS+PDT vs. IGS. (C) Kaplan-Meiersurvival curves of mice from the three experimental groups. IGS extendedthe animal survival significantly as compared with WLS (♦, P=0.0317).The survival was further prolonged by PDT.*, P=0.0008, IGS+PDT vs. WLS;#, P=0.0008 IGS+PDT vs. IGS.

FIG. 5 illustrates a synthesis scheme for Pc-Methacrylate that includesthe reaction of silicon phthalocyanine dihydroxide and3-methacryloxypropyldimethylchlorosilane in pyridine.

FIGS. 6 (A-B) illustrate (A) UV-VIS absorption profile and chemicalstructure of Pc4 (inset) and (B) Pc-Methacrylate (inset).

FIG. 7 is a graphical illustration of an electrospray ionization massspectrometry (ESI-MS) spectrum of Pc-methacrylate. (m/z): [M−OH]+calculated for M as C₄₁H₃₃N₈O₃Si₂, 741.22; found, 741.4.

FIGS. 8 (A-B) are graphs illustrating (A) decomposition of DPBF in thepresence of Pc-Methacrylate suggesting the generation of cytotoxicsinglet oxygen (¹O₂) species. (B) Comparison of the performance of Pc4and Pc-Methacrylate in generating ¹O₂ species.

FIG. 9 illustrates a plethora of exemplary PDT material of differentcomposition, structure, function and architecture that can besynthesized using polymerized Pc-Methacrylate.

FIGS. 10 (A-B) are synthesis schemes of (A) conjugation ofPc-Methacrylate to PSMA-Cys and (B) Conjugation of a RAFT-CTA to PSMA-Iand subsequent polymerization of Pc-Methacrylate and any polymer ofchoice.

DETAILED DESCRIPTION

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of theapplication.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbon groupscovalently linked by an oxygen. Accordingly, the substituent of an alkylthat renders that alkyl an ether is or resembles an alkoxy.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. Aryl groups include benzene, phenol,aniline, and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. Heteroaryl groupsinclude, for example, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the framework. It will beunderstood that “substitution” or “substituted with” includes theimplicit proviso that such substitution is in accordance with permittedvalence of the substituted atom and the substituent, and that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of thisinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms.Substituents can include, for example, a halogen, a hydroxyl, a carbonyl(such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), analkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate.

Substituents on fused ring structures can be peripheral ornon-peripheral substituents. A non-peripheral substituent, as definedherein, is a substituent which is adjacent (i.e., a) to the point offusion between an outer phenyl ring and an inner pyrrole ring, as foundin phthalocyanine compounds as exemplified by Formula (I) herein. Asubstituent is peripheral, on the other hand, when it is not anon-peripheral substituent. For example, in Formula I provided herein,the substituents R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are peripheralsubstituents.

As used herein, the term “targeting moiety” can refer to a molecule ormolecules that are able to bind to and complex with a biomarker. Theterm can also refer to a functional group that serves to target ordirect a compound described herein to a particular location, cell type,diseased tissue, or association. In general, a “targeting moiety” can bedirected against a biomarker.

As used herein, the term “molecular signature” can refer to a uniqueexpression pattern of one or more biomarkers (e.g., gene(s) orprotein(s)) of a cell.

As used herein, the term “neoplastic disorder” can refer to a diseasestate in a subject in which there are cells and/or tissues whichproliferate abnormally. Neoplastic disorders can include, but are notlimited to, cancers, sarcomas, tumors, leukemias, lymphomas, and thelike.

As used herein, the term “neoplastic cell” can refer to a cell thatshows aberrant cell growth, such as increased, uncontrolled cell growth.A neoplastic cell can be a hyperplastic cell, a cell from a cell linethat shows a lack of contact inhibition when grown in vitro, a tumorcell, or a cancer cell that is capable of metastasis in vivo.Alternatively, a neoplastic cell can be termed a “cancer cell.”Non-limiting examples of cancer cells can include melanoma, breastcancer, ovarian cancer, prostate cancer, sarcoma, leukemicretinoblastoma, hepatoma, myeloma, glioma, mesothelioma, carcinoma,leukemia, lymphoma, Hodgkin lymphoma, Non-Hodgkin lymphoma,promyelocytic leukemia, lymphoblastoma, thymoma, lymphoma cells,melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells,hepatoma cells, myeloma cells, glioma cells, mesothelioma cells, andcarcinoma cells.

As used herein, the term “tumor” can refer to an abnormal mass orpopulation of cells that result from excessive cell division, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition. As used herein, the terms “treating” or “treatment” of acancer can refer to executing a treatment protocol to eradicate at leastone cancer cell. Thus, “treating” or “treatment” does not requirecomplete eradication of cancer cells.

“PSMA” refers to Prostate Specific Membrane Antigen, a potentialcarcinoma marker that has been hypothesized to serve as a target forimaging and cytotoxic treatment modalities for cancer.

As used herein, the term “subject” can refer to any animal, including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish (e.g., zebrafish)), non-human primates,ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines,canines, felines, aves, etc.), which is to be the recipient of aparticular treatment.

Embodiments described herein relate to polymerizable phthalocyaninecompounds and prostate specific membrane antigen (PSMA)-targetedconjugates thereof for use in the detection and treatment of PSMAexpressing cancers and particularly for use in compositions and methodsof treating PSMA expressing cancers using photodynamic therapy (PDT).

The polymerizable phthalocyanine compounds are based on functionalanalogs of the PDT photosensitizing compound Pc4 that have been modifiedto include a methacrylate moiety. The addition of the methacrylatemoiety allows for the synthesis of polymers incorporating phthalocyaninecompounds. The methacrylate moiety also allows for conjugation ofphthalocyanine compounds to cysteine-containing peptide/proteins due tothe reactivity of the methacrylate double bond.

In some embodiments, the polymerizable phthalocyanine compound can havethe following formula (I):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and

R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, carboxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio, C₁₋₆hydroxyalkyl, C₁₋₆alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In certain embodiments, R¹-R¹⁶ of the compound of formula (I) areindependently selected from the group consisting of hydrogen, halogen,nitro, cyano, hydroxyl, thiol, amino, and methyl, while in otherembodiments R¹-R¹⁶ are all hydrogen.

In other embodiments, the polymerizable phthalocyanine compound can havethe formula (II):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ areeach independently selected from the group consisting of hydrogen,halogen, nitro, cyano, hydroxyl, thiol, amino, carboxy, aryl,heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆ alkylamino, C₁₋₆ thioalkyl,C₁₋₆alkylthio, C₁₋₆hydroxyalkyl, C₁₋₆ alkyloxycarbonyl, C₁₋₆alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In still other embodiments, the polymerizable phthalocyanine compoundcan have the formula (III):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and

R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, carboxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio, C₁₋₆hydroxyalkyl, C₁₋₆alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In some embodiments, R¹-R¹⁶ are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.

In certain embodiments, the polymerizable phthalocyanine compound canhave the formula

or a pharmaceutically acceptable salt thereof.

In an exemplary embodiment, a one-step synthetic process for producing aPc-Methacrylate phthalocyanine compound in accordance with oneembodiment is illustrated in FIG. 5 where silicon phthalocyaninedihydroxide and 3-methacryloxypropyldimethylchlorosilane are reacted inpyridine to form Pc-Methacrylate.

The reactivity of the methacrylate moiety of the polymerizablephthalocyanine compounds described herein allows for polymerization ofthe phthalocyanine compounds. Therefore, in some embodiments, a polymercomposition can include one or more of the polymerizable phthalocyaninecompounds described herein. The molecular weight, composition, structureand architecture of the polymer can be formulated using a variety ofwell-known polymer chemistry techniques. In particular embodiments,living radical polymerization (ATRP) and/or reversible-additionfragmentation chain transfer (RAFT) can be used to formulate a polymercomposition including one or more of the polymerizable phthalocyaninecompounds described herein.

A polymer including a polymerizable phthalocyanine compound describedherein can be formulated as a homopolymer. In some embodiments, one ormore of the polymerizable phthalocyanine compounds can be included in acopolymer. Copolymers synthesized using a polymerizable phthalocyaninecompound described herein can include, but are not limited to,alternating copolymers, block-copolymers, random-copolymers, brushcopolymers, comb-copolymers, graft copolymers, and dendrimer-starcopolymers. In an exemplary embodiment, a block copolymer can include ablock of a polymerizable phthalocyanine compound described herein and amonomer block having a multinuclear imaging agent moiety which allowsfor a material with MRI and PDT dual functionality. In certainembodiments, the monomer block having a multinuclear imaging agentmoiety is a ¹⁹F fluorinated monomer block.

In some embodiments, polymerizable phthalocyanine compounds describedherein can be coupled or conjugated to a cysteine-containingpeptide/protein. Such coupling or conjugation can occur via a reactioninvolving the cysteine thiol group and the terminal double bond of themethacrylate moiety to form a phthalocyanine compound conjugate. Inparticular embodiments, conjugation of a polymerizable phthalocyaninecompound described herein to a cysteine-containing peptide/protein canbe achieved using thiol-Michael addition click reaction. Additionalreactions for coupling or conjugating a polymerizable phthalocyaninecompound described herein to peptide/protein can include well-knownphoto, thermal and chemical crosslinking reactions involving doublebonds.

In some embodiments, the polymerizable phthalocyanine compound can becoupled or conjugated to at least one cysteine-containing targetingmoiety to target and/or adhere the phthalocyanine compound conjugate toa cell or tissue of interest. In some embodiments, the cell or tissue ofinterest can include a cancer cell or tumor tissue. The targetedphthalocyanine compound conjugates can target and transiently interactwith, bind to, and/or couple with a cancer cell, such as a prostatecancer cell, and once interacting with, bound to, or coupled to thetargeted cell or tissue advantageously facilitate delivery of thephthalocyanine compound within a cell by, for example, receptor mediatedendocytosis. The targeted conjugate including the polymerizablephthalocyanine compound and the targeting moiety can be formulated in apharmaceutically acceptable composition and administered to a subjectfor diagnostic, therapeutic, and/or theranostic applications.

The cysteine-containing targeting moiety coupled or conjugated to apolymerizable phthalocyanine compound described herein can include anymolecule, or complex of molecules, which is/are capable of interactingwith an intracellular, cell surface, or extracellular biomarker of thecell. The biomarker can include, for example, a cellular protease, akinase, a protein, a cell surface receptor, a lipid, and/or fatty acid.Other examples of biomarkers that the cysteine-containing targetingmoiety can interact with include molecules associated with a particulardisease. For example, the biomarkers can include cell surface receptorsimplicated in cancer, such as prostate specific membrane antigen (PSMA),CA-125 receptor, epidermal growth factor receptor, and transferrinreceptor. The cysteine-containing targeting moiety can interact with thebiomarkers through non-covalent binding, covalent binding, hydrogenbinding, van der Waals forces, ionic bonds, hydrophobic interactions,electrostatic interaction, and/or combinations thereof.

In some embodiments, the cysteine-containing targeting moiety maycomprise a ligand molecule, including, for example, ligands whichnaturally recognize a specific desired receptor of a target cell. Suchligand molecules include ligands that have been modified to increasetheir specificity of interaction with a target receptor, ligands thathave been modified to interact with a desired receptor not naturallyrecognized by the ligand, and fragments of such ligands. By way ofexample, where the cell targeted is a prostate cancer cell, thecysteine-containing targeting moiety can comprise a PSMA ligand.

Pathological studies indicate that PSMA is expressed by virtually allprostate cancers, and its expression is further increased in poorlydifferentiated, metastatic, and hormone-refractory carcinomas. HigherPSMA expression is also found in cancer cells from castration-resistantprostate cancer patients. Increased PSMA expression is reported tocorrelate with the risk of early prostate cancer recurrence afterradical prostatectomy. In addition to being overexpressed in prostatecancer (PCa), PSMA is also expressed in the neovasculature of neoplasmsincluding but not limited to conventional (clear cell) renal carcinoma,transitional cell carcinoma of the urinary bladder, testicular embryonalcarcinoma, colonic adenocarcinoma, neuroendocrine carcinoma,glioblastoma multiforme, malignant melanoma, pancreatic ductalcarcinoma, non-small cell lung carcinoma, soft tissue carcinoma, breastcarcinoma, and prostatic adenocarcinoma.

Therefore, in certain embodiments, the cysteine-containing targetingmoiety coupled or conjugated to a polymerizable phthalocyanine compounddescribed herein can include a highly negatively charged PSMA ligand(e.g., PSMA-1) having a terminal cysteine residue that allows forcoupling of the cysteine thiol group to the double bond ofPc-methacrylate, thereby forming a PSMA-targeted phthalocyanine compoundconjugate. In an exemplary embodiment, a cysteine-containing PSMAtargeting moiety can be directly coupled or conjugated to the site ofthe methacrylate terminal double bond of a Pc-Methacrylate compounddescribed herein via a thiol-Michael addition click reaction to producea PSMA-targeted theranostic PDT agent for the treatment of cancer (seee.g., FIG. 10A).

In certain embodiments, the PSMA-targeted phthalocyanine compoundconjugate can have the formula (V):

or a pharmaceutically acceptable salt thereof;

wherein m is 1-5, and wherein Y is a PSMA targeting moiety having acysteine residue, and wherein the PSMA targeting moiety targets thecomposition to a PSMA expressing cancer cell;

R¹, R⁴, R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, and methyl; and

R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are each independently selectedfrom the group consisting of hydrogen, halogen, nitro, cyano, hydroxyl,thiol, amino, carboxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆ alkoxy, C₁₋₆ acyl, C₁₋₆alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆ aminoalkyl, C₁₋₆alkylamino, C₁₋₆ thioalkyl, C₁₋₆ alkylthio, C₁₋₆ hydroxyalkyl, C₁₋₆alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, and C₁₋₆ alkylcarbonylamino.

In certain embodiments, R¹-R¹⁶ of the compound of formula (V) areindependently selected from the group consisting of hydrogen, halogen,nitro, cyano, hydroxyl, thiol, amino, and methyl, while in otherembodiments R¹-R¹⁶ are all hydrogen.

In other embodiments, the PSMA-targeted phthalocyanine compoundconjugate can have the formula (VI):

or a pharmaceutically acceptable salt thereof;

wherein Y is a PSMA targeting moiety having a cysteine residue, andwherein the PSMA targeting moiety targets the composition to a PSMAexpressing cancer cell.

In an exemplary embodiment, the PSMA-targeted phthalocyanine compoundconjugate can have the formula (VII):

or a pharmaceutically acceptable salt thereof.

PSMA-targeted phthalocyanine compound conjugates described herein havebeen found to be an effective alternative to PSMA-targeted Pc4 intargeted bioimaging of PSMA expressing cancer cells and visualization oftumor margins, intraoperative guided surgery (IGS) and/or targeted PDTof cancer in a subject. Without being bound by theory, it is believedthat the PSMA-targeted phthalocyanine compounds described herein canexploit the expression of the distinct biochemical marker, PSMA, foundfor example on the surface of PSMA expressing cancer cells, thussignificantly reducing both off-target effects and toxicity.

Therefore, additional aspects relate to the administration of apharmaceutical composition including a PSMA-targeted phthalocyaninecompound described herein to a subject for diagnostic, therapeutic,and/or theranostic applications.

In accordance with the method described herein, pharmaceuticalcompositions including a PSMA-targeted phthalocyanine compound describedherein can be administered to a subject in need thereof to detect and/ortreat a PSMA expressing cancer in subject. In some embodiments, the PSMAexpressing cancer is a tumor. The tumor can include a solid tumor, suchas a solid carcinoma, sarcoma or lymphoma, and/or an aggregate ofneoplastic cells. The tumor can include both cancerous and pre-cancerouscells. Exemplary PSMA expressing cancers treated in accordance with amethod described herein can include renal carcinoma, transitional cellcarcinoma of the urinary bladder, testicular embryonal carcinoma,colonic adenocarcinoma, neuroendocrine carcinoma, glioblastomamultiforme, malignant melanoma, pancreatic ductal carcinoma, non-smallcell lung carcinoma, soft tissue carcinoma, breast carcinoma, andprostatic adenocarcinoma. In certain embodiments, the PSMA cancer is ametastatic prostate cancer.

The PSMA-targeted phthalocyanine compound provided in a pharmaceuticalcomposition can be formulated in a pharmaceutically acceptable carrier.“Pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ or portion of the body, to another organ or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: sugars, such as lactose, glucose, andsucrose; starches, such as corn starch and potato starch; cellulose, andits derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil, and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

In some embodiments, aqueous-based or oil-based pharmaceuticallyacceptable carriers can be used. An aqueous-based pharmaceuticallyacceptable carrier is a polar solution primarily consisting of water,and including solutions such as pyrogen-free water, isotonic saline,Ringer's solution, and phosphate buffer solutions. Oil-basedpharmaceutically acceptable carriers, on the other hand, are relativelynon-polar solutions consisting primarily of oils or other relativelynon-polar organic solvents. Examples of oil-based pharmaceuticallyacceptable carriers include various organic solvents, mineral oil,vegetable oil, and petrolatum.

In some embodiments, pharmaceutical compositions including thePSMA-targeted phthalocyanine compounds can be formulated for systemic ortopical administration. Systemic administration includes delivery of anaqueous solution, preferably a buffered aqueous solution, including aphthalocyanine compound or targeted conjugate thereof. Systemicformulations typically also include a dispersant. Systemicadministration is typically done parenterally (e.g., intravenously orintramuscularly). However, systemic administration can also be carriedout by oral administration. By way of example, pharmaceuticalcompositions including PSMA-targeted phthalocyanine compounds describedherein can be intravenously administered to a subject that is known toor suspected of having a PSMA expressing tumor.

Topical administration of PSMA-targeted phthalocyanine compounds can beaccomplished using various different formulations such as powders,sprays, ointments, pastes, creams, lotions, gels, solutions, or patches.The active component may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required. The ointments, pastes,creams, solutions, foams, lacquers, oils and gels may contain excipientsin addition to phthalocyanine(s). These formulations may contain aphthalocyanine salt within or on micro or nanoparticles, liposomes,beads, polymer matrices, sponges, osmotic pumps, or other structures.

PSMA-targeted phthalocyanine compounds can be formulated as ointments orcreams for topical administration. Ointments are homogeneous, semi-solidpreparations intended for external application to the skin or mucousmembranes. They are used as emollients or for the application of activeingredients to the skin for protective, therapeutic, or prophylacticpurposes and where a degree of occlusion is desired. Ointments can beformulated using hydrophobic, hydrophilic, or water-emulsifying bases toprovide preparations for various applications. Creams, on the otherhand, are semi-solid emulsions; i.e., a mixture of oil and water. Theyare divided into two types: oil-in-water creams that are composed ofsmall droplets of oil dispersed in a continuous aqueous phase, andwater-in-oil creams that are composed of small droplets of waterdispersed in a continuous oily phase.

PSMA-targeted phthalocyanine compounds can also be administered inpharmaceutical compositions by aerosol. This is accomplished bypreparing an aqueous aerosol, liposomal preparation, or solid particlescontaining the compound. A nonaqueous (e.g., fluorocarbon propellant)suspension could be used. Sonic nebulizers are preferred because theyminimize exposing the agent to shear, which can result in degradation ofthe compound. Ordinarily, an aqueous aerosol is made by formulating anaqueous solution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers.

PSMA-targeted phthalocyanine compounds can also be formulated fordelivery as a gel. Gel formulations comprising a PSMA-targetedphthalocyanine compound or salt thereof may be prepared according toU.S. Pat. No. 6,617,356 or U.S. Pat. No. 5,914,334, the disclosures ofwhich are incorporated herein in their entirety. In addition,PSMA-targeted phthalocyanine compound-containing gels can be dried toform films suitable for phthalocyanine administration.

Transdermal patches have the added advantage of providing controlleddelivery of a phthalocyanine to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of thephotosensitizer(s) into the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe agent in a polymer matrix or gel.

Pharmaceutical compositions including PSMA-targeted phthalocyaninecompounds can also be delivered transdermally using microneedles. Seefor example Arora et al., International Journal of Pharmaceutics, 364,pg. 227-236 (2008), which describes micro-scale devices for transdermaldrug delivery.

Delivery of pharmaceutical compositions including PSMA-targetedphthalocyanine compounds described herein across an epithelial,epidermal, serosal, or mucosal surface may be accomplished usingapplication of an electrical current and a charged solvent solution,such as iontophoresis.

PSMA-targeted phthalocyanine compound compositions can be formulated toallow delivery in sufficient amounts and for a period of time(s) to bedetectable, imaging effective and therapeutically effective. Single ormultiple administrations of the probe can be given.

A PSMA-targeted phthalocyanine compound composition described herein canbe administered to a subject in a detectable and/or imaging effectivequantity. A “detectable quantity” means that the amount of thedetectable compound that is administered is sufficient to enabledetection of binding of the PSMA-targeted phthalocyanine compound to thePSMA expressing cancer cell. An “imaging effective quantity” means thatthe amount of the detectable compound that is administered is sufficientto enable imaging of binding of the compound to the targeted PSMAexpressing cancer cells. For example, the imaging effective quantity canbe the amount sufficient to enable detection PSMA-targetedphthalocyanine compound bound to the PSMA expressing cancer followed byintraoperative imaging during image guided surgical resection of cancerin a subject. In certain embodiments, the therapeutically effectiveamount is the amount sufficient to enable the induction of cytotoxiceffects of the PSMA-targeted phthalocyanine compound using PDT onresidual cancer cells following detection and surgical resection of thecancer. In exemplary embodiments, a PSMA-targeted phthalocyaninecompound composition is administered at a dose of about 0.5 mg/kg to asubject for use in the detection of binding of the PSMA-targetedphthalocyanine compound to the PSMA expressing cancer cells, imageguided surgical resection of the detected cancer cells, and subsequentPDT treatment on residual cancer cells following surgical resection inaccordance with a method described herein.

Once administered to a subject, a pharmaceutical composition includingPSMA-targeted phthalocyanine compound bound to and/or complexed with thePSMA expressing cancer cells is detected to determine the presence,location, and/or distribution of PSMA expressing cancer cells or PSMAexpressing neovaculature of the cancer cells in an organ or body area ofa subject. The presence, location, and/or distribution of thePSMA-targeting moiety coupled to a detectable phthalocyanine compound inthe animal's tissue, e.g., prostate tissue, can be visualized (e.g.,with an in vivo imaging modality). In an exemplary embodiment, thepresence, location, and/or distribution of the PSMA-targetedphthalocyanine compound bound to and/or complexed with the PSMAexpressing cancer cells can be visualized about 24 hours after thepharmaceutical composition is administered to the subject.

The imaging modality can include one or a combination of known imagingtechniques capable of visualizing the PSMA-targeted phthalocyaninecompound. Examples of imaging modalities can include ultrasound (US),magnetic resonance imaging (MRI), nuclear magnetic resonance (NMR),computed topography (CT), electron spin resonance (ESR), nuclear medicalimaging, optical imaging, and positron emission topography (PET). Theimaging modality can then be operated to generate a visible image of thepresence, location, and/or distribution of PSMA expressing cancer cells.

“Distribution” as used herein is the spatial property of being scatteredabout over an area or volume. In this case, “the distribution of cancercells” is the spatial property of cancer cells being scattered aboutover an area or volume included in the animal's tissue, e.g., prostatetissue. The distribution of the PSMA-targeting moiety coupled to adetectable phthalocyanine compound may then be correlated with thepresence or absence of PSMA expressing cancer cells in the tissue. Adistribution may be dispositive for the presence or absence of a cancercells or may be combined with other factors and symptoms by one skilledin the art to positively detect the presence or absence of migrating ordispersing cancer cells, cancer metastases or define a tumor margin inthe subject. In one embodiment, pharmaceutical compositions including aPSMA-targeted phthalocyanine compound are administered to a subject toassess the distribution of targeted cancerous tumor cells in a subjectand correlate the distribution to a specific location.

Following detection of PSMA-targeted phthalocyanine compounds bound toand/or complexed with the PSMA expressing cancer cells, the detectedcancer is used to define a tumor margin and guide surgical resection ofthe cancer from the subject. We have previously shown that aPSMA-targeted phthalocyanine compound used as a PDT agent alone, i.e.,without prior surgery, is sufficient to eradicate primary tumors in asubject but in 100% of the cases, the tumors recurred. Where the tumorburden may be too large for PDT alone to effectively eradicate all tumorcells, it was shown that therapeutic methods including surgicalresection using a PSMA-targeted phthalocyanine compound prior to PDTreduces the tumor burden, thereby allowing for more effectiveeradication of cancer cells and significantly reduced tumor recurrenceand improved survival of the subject.

PSMA-targeted phthalocyanine compounds for use as PDT agents can enhancesurgical cancer resection during image-guided surgery (IGS) compared towhite light surgical resection. Surgeons routinely use stereotactictechniques and intra-operative MRI (iMRI) in surgical resections. Thisallows them to specifically identify and resect tissue from distinctregions of the tumor such as the tumor edge or tumor center. Frequently,they also resect regions of targeted tissue on the tumor margin that areoutside the tumor edge that appear to be grossly normal but areinfiltrated by dispersing tumor cells upon histological examination.

The PSMA-targeted phthalocyanine compounds described herein can be usedin intra-operative imaging (IOI) techniques to guide surgical resectionand eliminate the “educated guess” of the location of the tumor by thesurgeon. Previous studies have determined that more extensive surgicalresection improves patient survival. It has also been shown that aPSMA-targeted phthalocyanine compound is easily detectable at real-timeimaging exposures (i.e., at <67 ms) and thus capable of being used forreal-time IGS during urological surgery. Thus, it is anticipated thatthe PSMA-targeted phthalocyanine compound conjugate compositionsdescribed herein for use in image-guided surgery can increase patientsurvival rates especially when combined with subsequent PDT inaccordance with a method described herein.

In some embodiments, IGS can be performed real-time using an in vivoimaging system, such as an intraoperative near-infrared fluorescenceimaging system. In some embodiments, image guided surgery can beperformed using a FLARE (Fluorescence-Assisted Resection andExploration) intraoperative NIR fluorescence imaging system where thetargeted cancer is imaged at a wavelength of about 671 nm to about 705nm. In particular embodiments, the targeted PMSA expressing cancer isimaged during image guided surgical resection at about 700 nm.

Following surgical resection, residual PSMA expressing cancer cells thathave penetrated beyond the resection site may revert to a proliferativestate to produce a more aggressive recurrent tumor that continues todisperse into nonneoplastic tissue adjacent the resection site andbeyond. A therapeutic method described herein can be used to minimizecancer metastasis after a surgical resection procedure targeting PSMApositive cancer cells and/or tumor tissue using photodynamic therapy(PDT).

Therefore, a therapeutic method described herein can further include thestep of irradiating the PSMA-targeted phthalocyanine compounds bound toand/or complexed with the PSMA expressing cancer cells remaining in,and/or adjacent to, the surgical site to induce the cytotoxic effects ofthe phthalocyanine compound on residual cancer cells following surgicalresection of the cancer.

Methods for conducting PDT are known in the art. See for example ThierryPatrice. Photodynamic Therapy; Royal Society of Chemistry, 2004. PDT isa site specific treatment modality that requires the presence of aphotosensitizer, light, and adequate amounts of molecular oxygen todestroy targeted tumors (Grossweiner, Li, The science of phototherapy.Springer: The Netherlands, 2005). Upon illumination, a photoactivatedsensitizer transfers energy to molecular oxygen that leads to thegeneration of singlet oxygen (O²) and other reactive oxygen species(ROS), which initiate apoptosis and oxidative damage to cancer cells.Only the cells that are exposed simultaneously to the targeted PDTcompound (which is non-toxic in the dark) and light are destroyed whilesurrounding healthy, non-targeted and nonirradiated cells are sparedfrom photodamage. Furthermore, the fluorescence of the phthalocyaninecompound coupled to the PSMA targeting moiety enable simultaneousdiagnostic optical imaging that can be used to guide the PDT step of themethod of treating cancer described herein.

Following administration and detection/localization of PSMA-targetedphthalocyanine compounds, the targeted cancer cells can be exposed totherapeutic amount of light that causes cancer cell damage and/orsuppression of cancer cell growth. The light, which can activate the PDTtherapeutic agent can be delivered to the targeted cancer cells, usingfor example, semiconductor laser, dye laser, optical parametricoscillator or the like. It will be appreciated that any source light canbe used if the light excites the phthalocyanine compound bound orcomplexed with a PSMA expressing cancer cell.

In some embodiments, the surgical resection site can be irradiated usingvisible laser diodes to photoactivate the phthalocyanine compoundcoupled to the PSMA targeting moiety. For example, the surgicalresection site can be irradiated using visible laser diodes emitting at672 nm. In certain embodiments, the surgical resection site can beirradiated with an amount of radiation effective to inhibit tumorrecurrence in the subject. In a particular embodiment, the PDT step ofthe method of treating cancer described herein can include irradiatingthe resection site bed with a 672 nm laser for about 12.5 minutes withtotal radiant exposure of about 75 J/cm².

Following the PDT step of a method of treating cancer described herein,the resection site of a subject can be further imaged and irradiatedafter a period of time(s) to detect and ablate residual PSMA expressingcancer cells that may have survived previous irradiation. This optionalstep may or may not include additional administration of apharmaceutical composition including a PSMA-targeted phthalocyaninecompound. In an exemplary embodiment, a pharmaceutical compositionincluding a PSMA-targeted phthalocyanine compound described herein canbe administered to a subject in need thereof to provide image guidancefor prostate tumor resection and allow subsequent targeted PDT toeliminate unresectable or remaining cancer cells.

The specific process utilized to administer and detect the PSMA-targetedphthalocyanine compounds of the present invention, and the enhancedresults produced by the compounds when used for a combination of imageguided surgery and photodynamic therapy, are more particularly describedbelow in the following examples. The following examples are for thepurpose of illustration only and are not intended to limit the scope ofthe claims, which are appended hereto.

Example 1

In this Example, we demonstrate a targeted photodynamic agent that canbe used for both image-guided surgery (IGS) and adjuvant targetedphotodynamic therapy (PDT), allowing better visualization of tumormargins and elimination of residual tumor tissues. This study presents anew treatment option for patients with prostate cancer undergoingsurgery, which significantly improves tumor visualization anddiscrimination during surgery, including identification of cancer inlymph nodes.

Using a prostate specific membrane antigen (PSMA)-targeted PDT agent wepreviously developed, PSMA-1-Pc413, we showed that PSMA-1-Pc413selectively highlighted PSMA-expressing tumors, allowing IGS and morecomplete tumor resection compared with white light surgery. SubsequentPDT further reduced tumor recurrence and extended animal survivalsignificantly. This approach also enabled identification of tumor cellsin lymph nodes.

PSMA-1-Pc413 strongly emits near-IR (NIR) light at 678 nm, binds toPSMA-expressing cancer cells, and is able to destroy them whenirradiated by NIR light. Due to limitations in light penetration andirradiating all tumor cells, we postulated that it might be moreeffective to use PSMA-1-Pc413 as a theranostic combining IGS and PDT tofully ablate nonresected tumor tissues and/or cancer cells. Currently,there are very few examples of IGS followed by PDT, and most of thesestudies rely on nonspecific or nontargeted uptake of PDT agents into thetumor, e.g., 5-ALA for glioblastoma. No such study has been performed onprostate cancer. In this study, we used PSMA-targeted Pc413 for IGSfollowed by PDT (FIG. 1A) and found that PSMA-1-Pc413 was able tovisualize cancer, enable more complete surgery, and effectively destroyinvisible localized microscopic cancer cells by PDT.

Materials and Methods Cell Culture

Retrovirally transfected PSMA-positive PC3pip cells were obtained fromDr. Michel Sadelain (Memorial-Sloan Kettering Cancer Center, New York,N.Y.). CWR22vr1 cells were obtained from the ATCC. Cells were lastchecked by Western blot and flow sorted in 2018, and no geneticauthentication was performed. Mycoplasma test was last performed in2015. Cells were maintained in RPMI1640 medium with 10% FBS. PC3pipcells were transfected with GFP by lentivirus infection. Cells werediscarded after passage 6.

Detection of ROS In Vivo

Animal experiments were approved by the University Institutional AnimalCare and Use Committee (#150033). Six- to 8-week-old male athymic nudemice were implanted subcutaneously with 1×10⁶ of PC3pip on the rightdorsum. When tumor diameter reached 10 mm, PSMA-1-Pc413 (0.5 mg/kg) wasinjected i.v. via the tail vein. Twenty-four hours later, mice received100 nmol of ROSstar800cw (Li-Cor Biosciences) in PBS. Animals wereimaged 30 minutes later and then illuminated with 672 nm laser (AppliedOptronics Corp) with irradiance of 33.3 mW/cm2 for 25 minutes (totalradiant exposure of 150 J/cm2). Fluorescence imaging was performed on aMaestro in vivo Imaging system (Perkin-Elmer): yellow filter forPSMA-1-Pc413 signal (excitation 575-605 nm, emission filter 645 nmlongpass); deep red filter for ROSstar800cw (excitation 671-705 nm,emission filter 750 nm longpass). Experiments were repeated in 3 mice.

Detection Sensitivity for PSMA-1-Pc413

PSMA-1-Pc413 and Forte700NHSester (Curadel LLC) were serially diluted inmouse serum and taken up by microcapillary tubes. The tubes wereinserted to a cap device with 10 mL sample volume window (Curadel LLC)and imaged at 700 nm (Curadel RP1 Fluorescence Image System) using 100%power and varying exposure. Signals from each capillary tube werequantified using Curadel software.

In Vivo Image of PSMA-1-Pc413 in Orthotopic Mice Model

To establish an orthotopic prostate cancer model, 6- to 8-week oldathymic nude mice were first anesthetized by i.p. injection of 50 mg/kgketamine/xylazine. A transverse incision was made in the lower abdomento expose the prostate. Ten microliters of PC3pipGFP cells (5×10⁵) inPBS were injected into the dorsal lateral prostate gland. The incisionin the abdominal wall was then closed. After about 4 weeks (tumordiameter approximately 1 cm), mice receive 0.5 mg/kg PSMA-1-Pc413 andwere imaged 24 hours later by a Maestro imaging device (yellow filter:PSMA-1-Pc413, excitation 575-605 nm, emission filter 645 nm longpass;and blue filter: GFP, excitation 445-490 nm, emission filter 515 nm longpass). Mice were then euthanized. After the primary tumor was removed toexpose the lymph nodes (LN) buried behind the primary tumor, the mousewas again imaged. The resected primary tumor and lymph nodes were fixedin 10% formalin, paraffin embedded, sectioned, and slides prepared. Oneset of the slides was subjected to hematoxylin and eosin staining, andthe adjacent set was observed under a Leica DM4000B fluorescencemicroscope (Leica Microsystem Inc.) to visualize GFP and PSMA-1-Pc413fluorescence. Experiments were repeated in 5 mice.

Intramuscle Prostate Tumor Xenograft Model

PSMA-positive PC3pipGFP cells (1×10⁶) were injected into the muscle ofthe right leg of 6- to 8-week-old male athymic nude mice. Tumor growth(GFP signal) was monitored twice a week using the Maestro imaging deviceand calipers. When tumors reached 200 mm³, further studies wereperformed.

In Vivo Surgery and PDT Treatment of Intramuscle PC3pipGFP Tumors

Mice were randomly divided into three groups: WLS, IGS, and IGS,followed by PDT (IGS+PDT; FIG. 1A). All mice received 0.5 mg/kgPSMA-1-Pc413 via tail vein injection. Surgery was performed at 24 hoursafter injection for peak accumulation of PSMA-1-Pc413. Before surgery,preimages for both GFP and PSMA-1-Pc413 were obtained. WLS was performedunder room light. For IGS, tumors were removed under the guidance ofCuradel RP1 at an exposure (50 ms), which allowed real-time imaging.After surgery, mice were imaged again by Maestro, and then the woundswere sutured for WLS group and IGS group. For the IGS+PDT group ofanimals, the resection bed was irradiated with 672 nm laser (AppliedOptronics Corp) for 12.5 minutes with total radiant exposure of 75J/cm2. Light was delivered through a GRIN-lens-terminate multimode fiber(OZ Optics) and was adjusted to cover all the surgical area (˜1.0-1.5 cmin diameter). After PDT, the mice were imaged by Maestro, and the woundswere sutured. Tumor growth was then monitored by Maestro every other dayfor 80 days. Mice were terminated when the tumor reached the size of 1.5cm.

Statistical Analysis

The Student t test was used to compare Maestro signals in differenttreatment groups. Tumor recurrence and Kaplan-Meier survival data wereanalyzed by SAS9.4 using exact pairwise Wilcoxon rank-sum test. A Pvalue<0.05 was considered statistically significant for all comparisons.

Results

PSMA-1-Pc413 Generated ROS after PDT

To confirm that PSMA-1-Pc413 generates ROS in vivo, its proposedmechanism of action, we used ROSstar800cw conversion into fluorescentcyanine to detect ROS. Before PDT, PSMA-1-Pc413 fluorescent signal wasmainly observed in PC3pip tumors (FIG. 1B, top plot, 2nd and 3rdcolumns) with minimal ROSstar800cw fluorescence observed (FIG. 1B,bottom plot, 2nd and 3rd columns). After PDT, there was a dramaticincrease in ROSstar800cw fluorescence, and little PSMA-1-Pc413 signalwas observed due to photo activation (FIG. 1B, 4th column). Ex vivoimages of the bisected tumor showed that ROS was present throughout thetumor tissues (FIG. 1B, 5^(th) column). To assess IGS sensitivity (i.e.,detection limit), we compared PSMA-1-Pc413 fluorescence with Forte700NHS ester, a typical agent used to derive IGS probes. At 50 ms exposuretime, the fluorescent signal from PSMA-1-Pc413 was about 2-fold higherthan that from Forte700. At the highest exposure, 10 femtomol ofPSMA-1-Pc413 was able to be detected, suggesting that PSMA-1-Pc413 wassuitable for IGS when imaged using the Curadel RP1 camera system.

Detection of Primary Tumors and LN Metastasis

Most deaths from cancer are a result of metastasis. Detection of LNmetastases is of major prognostic significance for many cancers. Usingan orthotopic human prostate cancer mouse model, reported to develop LNmetastases, we tested if PSMA-1-Pc413 has the ability to detect LNmetastases. Twenty-four hours after PSMA-1-Pc413 administration, micebearing orthotopic PC3pipGFP tumors displayed coincident GFP and PC413fluorescent signals in the primary tumor (FIG. 2A). Removal of theprimary tumor revealed enlarged iliac LNs as reported by others.Fluorescence imaging demonstrated coincidence of GFP and PSMA-1-Pc413fluorescence in the LNs (FIG. 2A). Pathologic analysis demonstrated thatboth the prostate gland and LNs contained cancer and that the GFP andPSMA-1-Pc413 signals were highly correlated (FIG. 2B), demonstratingthat PSMA-1-Pc413 could detect both primary prostate tumor and LNmetastases.

IGS Using PSMA-1-Pc413

To assess the potential utility of PSMA-1-Pc413 to aid tumor resectionand ablation of remaining tumors/cancer cells, we compared animalsreceiving WLS, IGS, and IGS+PDT (FIG. 1A). Because mouse orthotopicprostate cancer models are not suitable for surgery, we developed anintramuscle tumor model. The muscular injection site resulted in tumorsthat were less encapsulated than subcutaneous xenografts, had sometissue infiltration, causing more challenges for surgery. We alsodemonstrated microdispersions of the cells away from the tumor massusing Cryo-imaging Twenty-four hours after injection probe, coincidentfluorescent signal was observed in PC3pipGFP tumors for both GFP andPSMA-1-Pc413 (FIGS. 3A and B, 2^(nd) column). Tissue distribution at 24hours after injection of PSMA-1-Pc413 showed highest uptake in theliver, followed by PC3pip tumor, then kidneys with washout occurring forthe organs by 96 hours. Prior to surgery, the GFP and PSMA-1-Pc413signals had similar intensities in all groups (FIGS. 3C and D, left).Postoperative imaging showed fluorescence remaining in the surgicalfield for WLS (FIG. 3A, 3 rd column) with both GFP and PSMA-1-Pc413, andPSMA-1-Pc413 signal was detectable using the Curadel camera (FIG. 3A,bottom row). When IGS was performed, high fluorescence was observed inthe tumor, which helped identify tumor tissues (FIG. 3B, bottom row).Minimal remaining fluorescence (GFP or PSMA-1-Pc413) was observed inmice that underwent IGS (FIG. 3B, 3 rd column). Comparison of averagefluorescent signals showed that both the GFP and PSMA-1-Pc413 signals(FIGS. 3C and D, middle) were significantly lower in IGS and IGS+PDTgroups than those in the WLS group (P<0.05). These results show that IGSwas able to highlight tumor tissue not visible during WLS, resulting inmore complete tumor removal. Sequential PDT treatment of the IGSsurgical wound led to significant reduction in PSMA-1-Pc413 fluorescentsignal, indicating PDT was activated in the surgical bed (FIG. 3B, 4thcolumn; FIG. 3D, right). GFP signal in the IGS wound was notsignificantly reduced after PDT, (FIG. 3C, right). To demonstrate thatfluorescence was associated with tumor tissues, a total of 92 pieces ofboth fluorescing and nonfluorescing tissues resected during IGS andIGS+PDT procedures were collected and underwent blinded pathologicanalysis. Among the 92 samples, 22 did not show any fluorescence andwere pathologically negative for cancer (true negatives); 68 showedfluorescence and were positive for cancer (true positives); 2 showedfluorescence, but were not cancer (false positives); and there were nofalse negatives. Analysis demonstrated sensitivity and specificity of100% and 91.7%, respectively. Histologic and fluorescence microscopicexamination of the resected tumor tissue showed that PSMA-1-Pc413 signalcorresponded well with the tumor tissue, was able to delineate theborderline between cancer tissue and normal tissue, and was able toidentify cancer cells that invaded into normal tissue.

Taken together, these data suggest that the technology may provideprecise guidance for surgery. To further confirm that IGS will achievemore complete tumor resection, a separate set of surgeries wasperformed, after which, the mouse legs with surgical wounds werecollected, sectioned, and examined by pathology. It was found that all 5mice in WLS group had remaining tumor, 3 of 5 mice in IGS group hadtumor, and all 4 mice in IGS+PDT group were tumor free. No damage tonormal tissues was observed in the IGS+PDT wound beds following PDT

PDT in Combination with IGS Reduced Tumor Recurrence

After surgery, we next monitored tumor recurrence (GFP signal) andanimal survival to evaluate overall response to the theranosticapproach. In the WLS group, residual GFP signal was observed aftersurgery and rapid tumor growth resulted in strong GFP signal by day 10(FIG. 4A, 1^(st) row). Both IGS and IGS+PDT mice showed no/minimal GFPsignal after surgery (FIGS. 3C and 4A). The IGS mice started to show GFPsignal on day 36 after surgery (FIGS. 4A and B), but no significantdifference was observed in tumor recurrence between WLS and IGS mice(P=0.2222). In contrast, only one mouse in the IGS+PDT group showedtumor recurrence on day 80 after surgery, and the other 7 were tumorfree; significant differences were observed between IGS+PDT versus IGS(P=0.00084), and IGS+PDT versus WLS (P=0.0008; FIGS. 4A and B). IGSsignificantly extended animal survival compared with WLS (P=0.0317), andIGS+PDT further prolonged animal survival to >80 days (P=0.0008 comparedwith WLS and P=0.0008 compared with IGS, FIG. 4C). No adverse effects ordelay in healing was observed in IGS+PDT animals as compared with WLSand IGS animals. We also performed studies using mice bearing prostatetumors derived from human CWR22rv1 cells, which spontaneouslyoverexpress PSMA at a level only 1/12 of that in PC3pip cells. Despitethe lower PSMA expression, the level of PSMA-1-Pc413 uptake in CWR22rv1cells was about 50% of that in the PC3pip tumors and PDT effectivelyinhibited tumor growth without surgical intervention. When CWR22rv1tumors were implanted into mouse flank muscles, PSMA-1-Pc413 fluorescentsignal was clearly visible and suitable for IGS. IGS+PDT againsignificantly delayed tumor recurrence and extended animal survival ascompared with WLS and IGS groups.

PDT has been used for selective identification and treatment of cancers.Postsurgical photoimmunotherapy (PIT) has shown inhibition of tumorrecurrence after surgical resection of pancreatic and head and neckcancer models. However, the ability of PIT to aid IGS in the treatmentof prostate cancer has not been explored. Further, these antibody agentsare significantly more expensive to make than the small urea/peptidebased agent used here, and antibody's longer circulation time could leadto off target accumulation. Although there are a few examples of IGSfollowed by nontargeted PDT, there are none for prostate cancer, whichhas 20% to 40% local recurrence that is associated with incompletesurgery. Even in the few examples where IGS was followed by PDT forother cancers, the molecules are not as selective or designed to exploitdistinct biochemical biomarkers for the disease targeted and thereforelikely may have significant off-target effects and increased toxicity.We have developed a completely novel PSMA-targeted PDT agent to directlyexploit overexpression of a biomarker on the surface of most prostatecancers (>95% overexpression). We initially tested this agent as a PDTagent only, i.e., without prior surgery, and demonstrated that primarytumors (Tstage) could be eradicated, but in all cases recurred. Here, wetest the hypothesis that recurrence of primary tumors after PDTtreatment is not complete because the tumor burden is too large for PDTto effectively “get” all the tumor cells. We hypothesized that IGS willreduce tumor burden and, followed immediately by PDT, improve survival.We showed that PSMA-1-Pc413 is easily detectable at realtime imagingexposures (<67 ms) and can provide real-time IGS for urologicalsurgeons. It was noticed that tumor depths obscured some GFP signal whenimaged in vivo, but in all cases when tumors were excised, there was agood correlation between tumor GFP expression and PSMA-1-Pc413fluorescence.

Surgery is the main treatment option for primary prostate cancer. In ournovel approach, surgical resection was improved by PSMA-1-Pc413 IGS and,when immediately followed by PDT, significantly improved outcome. Thecombined approach resulted in a local tumor recurrence rate of only 1 of8 animals, first detectable 80 days after completion of the procedure,i.e., IGS+PDT (theranostic) cures prostate cancer 87.5% of the time.Cryo-image of the tumor-bearing mice showed microdispersion of cancercells away from the primary tumor mass, underscoring the prudence of thehypothesis and the importance of adjuvant PDT treatment. Pathologyshowed a good correlation between tumor remaining in the wound bed andtumor recurrence. IGS+PDT did not damage surrounding normal tissues.Pathology studies of resected tissues showed that the fluorescent tissuepieces resected during IGS were largely cancerous, achieving asensitivity of 100% and specificity of 91.7%, respectively. It isunclear why the specificity was only 91.7%, given the selectivity of thePSMA-1-Pc413 molecules and the selective expression of the receptor. Itis possible that the tissues that were false positive perhaps hadsignificant background fluorescence due to occasional wounds inflictedon the animals by each other. Even though PC3pip cells were engineeredto overexpress the PSMA receptor, we used them for these studies because(i) they grow much more rapidly in mice than other prostate cancer celllines and (ii) express levels of PSMA similar to many prostate cancercells lines that have not been engineered to overexpress PSMA. Werepeated studies with CWR22rv1 cells and demonstrated that the lowerexpression of PSMA in CWR22rv1 tumors does not significantly affect theutility of the approach to remove residual tumor tissue with PDTfollowing surgery. Interestingly, the Western blot measurements ofreceptor levels suggest that lower PSMA levels on CWR22rv1 cells canstill load cells with substantial level of the PSMA-1-PC413 molecule invivo. More studies are underway to determine the reason(s) for thesedifferences. Nevertheless, the lower levels of PSMA receptor levels aresuitable for IGS+PDT approach. It has been reported recently thatlow-level PSMA expression is enough for tumor targeting and imaging,which supported our findings. Further, PSMA-1-Pc413 was able to detectLN metastases (FIG. 2 ).

In conclusion, application of combined IGS and PDT technologies may beable to improve clinical treatment of prostate cancer for patients thatelect to undergo radical prostatectomy. It will improve cancer tissuevisualization and enable discrimination among cancerous, normal, neural,and muscle cells and tissues during surgery and has the ability to helpvisualize LN metastases. In particular, the PDT component of thedeveloped theranostic probe provides an adjuvant therapeutic approach todestroy unresectable tissues and/or missed cancer cells, reducing thefrequency of positive margins and cancer recurrence. The technology isnovel, innovative, and has great potential to be translated into theclinic benefiting patients with prostate cancer but will require outcomestudies in addition to decreasing positive tumor margins.

Example 2

In this example, we demonstrate the synthetization of a functionalanalog of the silicon phthalocyanine photosensitizing PDT agent, Pc4,based on a facile one-step synthetic reaction illustrated in FIG. 5 .With stoichiometric control, the reaction of silicon phthalocyaninedihydroxide and 3-methacryloxypropyldimethylchlorosilane in pyridineaffords the formation of Pc-Methacrylate (FIG. 6 b , inset). Thereaction product was purified by column chromatography with 1:10MeOH:DCM as the running solvent. Spectroscopic techniques such asabsorption (FIG. 6 ) and mass spectroscopy confirmed the formation ofthe desired product (FIG. 7 ). Interestingly, the as-synthesizedPc-Methacrylate displays a very similar absorption profile as that ofPc4.

A principal of photodynamic therapy is the ability of the PDT agent togenerate reactive oxygen species (ROS) mainly singlet oxygen (¹O₂) whenactivated by light. These generated products are highly reactive and canoxidize key cellular components leading to tumor ablation. To confirmthe ability of Pc-methacrylate to generate singlet oxygen, we performedthe photo-irradiation of Pc-methacrylate in the presence of a singletoxygen trap diphenylisobenzofuran (DPBF). When ¹O₂ is generated, DPBFphotodecomposes which can be conveniently monitored by UV-Vis absorptionspectroscopy. FIG. 8 a demonstrates the performance of Pc-methacrylatein generating ¹O₂ at different irradiation doses. This data confirmsthat the synthesized Pc-methacrylate compound is a promising moleculefor PDT application. In addition, it appears that the performance ofPc-methacrylate is similar to that of Pc4 (FIG. 8 b ). This data showsthat Pc-Methacrylate can be used as a small molecule alternative to Pc4.

In addition, the reactivity of the methacrylate moiety inPc-methacrylate makes it more attractive not only for polymerizationpurposes but also for the many chemistries afforded by the terminaldouble bond (FIG. 9 ). Regarding polymers, a material based onPc-Methacrylate can be designed in a polymeric form where moleculeweight, composition, structure and architecture can by formulated usinga variety of polymer chemistry techniques. This includes, but is notlimited to, living radical polymerization (ATRP) and reversible-additionfragmentation chain transfer (RAFT). Functional block-copolymers, randomcopolymers, graft copolymers and all other architectures may be easilyafforded by introducing the polymerizable molecule of choice. Forexample, a material with dual functionality based on block copolymercontaining Pc-Methacrylate block and fluorinated block may be used for(¹⁹F) MRI and PDT. On the other hand, we can take advantage of thereactivity of the double bond to several chemical moieties. For example,conjugation of Pc-Methacrylate by thiol-Michael addition click reactionon cysteine-containing peptide/protein may easily be achieved. Reactionsdescribed herein also include photo, thermal and chemical crosslinkingtechniques involving double bonds.

We have also conjugated Pc-Methacrylate to thiol-bearing PSMA-Cysteineas shown in the synthesis scheme depicted in FIG. 10A. We havepreviously developed PSMA-1-Pc413 and PSMA-1-IR700 as promisingtheranostic PDT agents for the treatment of prostate cancer. Thesynthesis of PSAM-Pc-Methacrylate will not only add to the library oftheranostic PDT agents but also provides a more stable therapeuticPSMA-targeted composition.

We have also synthesized RAFT-CTA bearing PSMA through an NHS-esterreaction with the terminal amine of PSMA-1 (FIG. 10B). This synthesisresults in a PMA-1-RAFT compound where monomers can be inserted in acontrolled fashion. For instance, PC-Methacrylate can be polymerizedresulting in a PC-block and subsequent polymerization of a monomer ofchoice, depending on the desired function, can be incorporated.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, we claim:
 1. A phthalocyanine compoundhaving the formula (I):

or a pharmaceutically acceptable salt thereof; wherein m is 1-5; R¹, R⁴,R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selected from thegroup consisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol,amino, and methyl; and R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are eachindependently selected from the group consisting of hydrogen, halogen,nitro, cyano, hydroxyl, thiol, amino, carboxy, aryl, heteroaryl,carbocyclyl, heterocyclyl, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆alkoxy, C₁₋₆ acyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆aminoalkyl, C₁₋₆ alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio,C₁₋₆hydroxyalkyl, C₁₋₆ alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, andC₁₋₆ alkylcarbonylamino.
 2. The phthalocyanine compound of claim 1,wherein R¹-R¹⁶ are independently selected from the group consisting ofhydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino, and methyl. 3.The phthalocyanine compound of claim 1, having the formula (II):

or a pharmaceutically acceptable salt thereof; wherein m is 1-5; R¹, R⁴,R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selected from thegroup consisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol,amino, and methyl; and R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are eachindependently selected from the group consisting of hydrogen, halogen,nitro, cyano, hydroxyl, thiol, amino, carboxy, aryl, heteroaryl,carbocyclyl, heterocyclyl, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆alkoxy, C₁₋₆ acyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆aminoalkyl, C₁₋₆ alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio,C₁₋₆hydroxyalkyl, C₁₋₆ alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, andC₁₋₆ alkylcarbonylamino.
 4. The phthalocyanine compound of claim 3,wherein R¹-R¹⁶ are independently selected from the group consisting ofhydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino, and methyl. 5.The phthalocyanine compound of claim 1 having the formula (III):

or a pharmaceutically acceptable salt thereof; wherein m is 1-5; R¹, R⁴,R⁵, R⁸, R⁹, R¹², R¹³, and R¹⁶ are each independently selected from thegroup consisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol,amino, and methyl; and R², R³, R⁶, R⁷, R¹⁰, R¹¹, R¹⁴, and R¹⁵ are eachindependently selected from the group consisting of hydrogen, halogen,nitro, cyano, hydroxyl, thiol, amino, carboxy, aryl, heteroaryl,carbocyclyl, heterocyclyl, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl, C₁₋₆alkoxy, C₁₋₆ acyl, C₁₋₆ alkylcarbonyloxy, C₁₋₆ carbocyclylalkyl, C₁₋₆aminoalkyl, C₁₋₆ alkylamino, C₁₋₆ thioalkyl, C₁₋₆alkylthio,C₁₋₆hydroxyalkyl, C₁₋₆ alkyloxycarbonyl, C₁₋₆ alkylaminocarbonyl, andC₁₋₆ alkylcarbonylamino.
 6. The phthalocyanine compound of claim 5,wherein R¹-R¹⁶ are independently selected from the group consisting ofhydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino, and methyl. 7.The phthalocyanine compound of claim 1, having the formula (IV):

or a pharmaceutically acceptable salt thereof.
 8. A pharmaceuticalcomposition for treating cancer in a subject, the composition comprisinga plurality of PSMA-targeted phthalocyanine compounds having theformula:

or a pharmaceutically acceptable salt thereof; wherein m is 1-5 and Y isa PSMA targeting moiety having a terminal cysteine residue, and whereinthe PSMA targeting moiety targets the composition to a PSMA expressingcancer cell.
 9. The pharmaceutical composition of claim 8, whereinR¹-R¹⁶ are independently selected from the group consisting of hydrogen,halogen, nitro, cyano, hydroxyl, thiol, amino, and methyl.
 10. Thepharmaceutical composition of claim 8, wherein the compounds have theformula (VI):

or a pharmaceutically acceptable salt thereof; wherein Y is a PSMAtargeting moiety having a terminal cysteine residue, and wherein thePSMA targeting moiety targets the composition to a PSMA expressingcancer cell.
 11. The pharmaceutical composition of claim 8, wherein thecompounds have the formula (VII):

or a pharmaceutically acceptable salt thereof
 12. The pharmaceuticalcomposition of claim 10, wherein the PSMA targeted phthalocyaninecompounds are formulated with a pharmaceutically acceptable carrier. 13.The pharmaceutical composition of claim 10, being formulated forsystemic administration.
 14. A method for treating a PSMA expressingcancer in a subject, the method comprising: (a) administering to thesubject with PSMA expressing cancer a therapeutically effective amountof a pharmaceutical composition, the pharmaceutical compositionincluding a plurality of PSMA-targeted phthalocyanine compounds havingthe formula (V):

 or a pharmaceutically acceptable salt thereof; wherein m is 1-5 and Yis a PSMA targeting moiety having a terminal cysteine residue, andwherein the PSMA targeting moiety targets the composition to a PSMAexpressing cancer cell, and pharmaceutically acceptable salts thereof;(b) detecting PSMA-targeted phthalocyanine compounds bound to and/orcomplexed with the cancer cells to determine the location and/ordistribution of the cancer cells in the subject; and (c) irradiating thedetected PSMA-targeted phthalocyanine compounds, thereby inducing thecytotoxic effects of the phthalocyanine compound on the cancer cells.15. The method of claim 14, wherein R¹-R¹⁶ of the at least onephthalocyanine compound are independently selected from the groupconsisting of hydrogen, halogen, nitro, cyano, hydroxyl, thiol, amino,and methyl.
 16. The method of claim 14, further comprising the steps of:surgically resecting the cancer in the subject, wherein the detectedPSMA-targeted phthalocyanine compound conjugates bound to and/orcomplexed with the cancer cells guide surgical resection of the cancer;and irradiating the PSMA-targeted phthalocyanine compound at the site ofsurgical resection, thereby inducing the cytotoxic effects of thephthalocyanine compound on residual cancer cells following surgicalresection.
 17. The method of claim 16, wherein the surgical resectionsite is irradiated with an amount of radiation effective to inhibittumor recurrence in the subject.
 18. The method of claim 16, whereinintra-operative imaging (IOI) of the PSMA-targeted phthalocyaninecompound conjugate bound to and/or complexed with the cancer cellsdefines a tumor margin in the subject to guide surgical resection of thecancer.
 19. The method of claim 14, the PSMA-targeted phthalocyaninecompound conjugate having the formula (VI):

or a pharmaceutically acceptable salt thereof; wherein Y is a PSMAtargeting moiety having a terminal cysteine residue, and wherein thePSMA targeting moiety targets the composition to a PSMA expressingcancer cell.
 20. The method of claim 14, wherein the PSMA expressingcancer is selected from the group consisting of renal carcinoma,transitional cell carcinoma of the urinary bladder, testicular embryonalcarcinoma, colonic adenocarcinoma, neuroendocrine carcinoma, gliobastomamultiforme, malignant melanoma, pancreatic ductal carcinoma, non-smallcell lung carcinoma, soft tissue carcinoma, breast carcinoma, andprostatic adenocarcinoma.